Methods and kits for detection of active malignancy

Abstract

Various embodiments of methods and kits are disclosed for detection and/or diagnosis of cancer (e.g., active malignancy) in a subject patient by analyzing a first sample of the subject patient's albumin-containing extracellular fluid (e.g., blood serum). Some embodiments comprise analyzing a second sample of the subject patient's albumin-containing extracellular fluid obtained between 2 and 90 days (e.g., between 5 and 30 days) after the first sample.

The invention claimed is: 
     
       1. A method for detecting active malignancy in a subject patient, comprising:
 mixing a labeled hydrophobic probe capable of binding to specific and non-specific hydrophobic binding sites of serum albumin with an aliquot of a first sample of a subject patient's serum albumin-containing extracellular fluid, the amount of probe such that the molar ratio of the probe to the serum albumin is between 2 and 7, the probe optionally comprising two or more substances; 
 mixing a solvent with the aliquot of the first sample, the solvent such that when added to the aliquot and probe the solubility of the probe is increased in the aliquot, and the amount of solvent is sufficient to dissociate a portion of the probe from the serum albumin without causing significant dissociation of hydrophobic metabolites from the serum albumin, the solvent optionally comprising two or more substances; 
 analyzing the mixture of aliquot, probe, and solvent to determine for the first sample one or more binding parameters of the probe, where analyzing the mixture comprises:
 substantially excluding from the mixture a portion of the probe that is bound on lipoproteins and a portion of the probe that is aggregated into micelles; and 
 determining the one or more probe binding parameters of the sample from the remaining portion of the probe in the mixture; and 
 
 comparing the one or more binding parameters of the first sample to one or more control binding parameters of a probe binding to serum albumin in the extracellular fluid of at least one of: a cancer-free control patient and a control patient with an active malignancy. 
 
     
     
       2. The method of  claim 1 , further comprising:
 mixing a labeled hydrophobic probe capable of binding to specific and non-specific hydrophobic binding sites of serum albumin with an aliquot of a second sample of the subject patient's serum albumin-containing extracellular fluid taken from the subject patient between 2 and 90 days after the first sample, the amount of probe such that the molar ratio of the probe to the serum albumin is between 2 and 7, the probe optionally comprising two or more substances; 
 mixing a solvent with the aliquot of the second sample, the solvent such that when added to the aliquot and probe the solubility of the probe is increased in the aliquot, and the amount of solvent is sufficient to dissociate a portion of the probe from the serum albumin without causing significant dissociation of hydrophobic metabolites from the serum albumin, the solvent optionally comprising two or more substances; 
 analyzing the mixture of aliquot, probe, and solvent to determine for the second sample one or more binding parameters of the probe, where analyzing the mixture comprises:
 substantially excluding from the mixture a portion of the probe that is bound on lipoproteins and a portion of the probe that is aggregated into micelles; and 
 determining the one or more probe binding parameters of the sample from the remaining portion of the probe in the mixture; and 
 
 comparing the one or more binding parameters of the second sample to one or more control binding parameters of a probe binding to serum albumin in the extracellular fluid of at least one of: a cancer-free control patient and a control patient with an active malignancy. 
 
     
     
       3. The method of  claim 2 , further comprising:
 diagnosing an active malignancy in the subject patient if:
 (i) the one or more binding parameters for each of the two samples are closer to the one or more control binding parameters for the at least one control patient with an active malignancy than to the one or more control binding parameters of the cancer-free control patient, and 
 (ii) the subject patient had no indication of acute disease or infection that occurred between the times of the deriving of the samples. 
 
 
     
     
       4. The method of  claim 2 , where the one or more probe binding parameters of each sample are determined from the portion of the probe bound to specific and non-specific hydrophobic binding sites of albumin, and the portion of unbound probe. 
     
     
       5. The method of  claim 2 , where the two samples are taken from the subject patient between 5 and 90 days apart, between 2 and 30 days apart, or between 5 and 30 days apart. 
     
     
       6. The method of  claim 2 , further comprising:
 verifying that the subject patient had no indication of acute diseases that occurred between the times at which the two samples were taken from the patient. 
 
     
     
       7. The method of  claim 2 , where analyzing each mixture comprises:
 measuring the concentrations of the probe bound on specific and non-specific hydrophobic sites of serum albumin, and the unbound probe in the mixture; and 
 deriving the one or more probe binding parameters from at least the concentrations of: the probe bound to specific and non-specific hydrophobic binding sites of albumin, and the unbound probe. 
 
     
     
       8. The method of  claim 2 , where the probe comprises an organic molecule having between 8 and 28 Carbon atoms, and optionally, where the probe comprises one or more of: a fatty acid, a long-chain fatty acid, 16-DOXYL-stearic acid, or a lysophospholipid. 
     
     
       9. The method of  claim 2 , where the amount of solvent mixed with the aliquot and probe is such that the solvent does not induce significant conformational changes of albumin. 
     
     
       10. The method of  claim 2 , where the amount of solvent mixed with the aliquot and probe is sufficient to increase the concentration of unbound probe in the mixture of the aliquot, the probe, and the solvent to at least 5 times greater than the concentration of unbound probe in a mixture of the aliquot and the probe without the solvent. 
     
     
       11. The method of  claim 2 , where the volume of the solvent mixed with the aliquot is less than 30% of the volume of the aliquot, and optionally less than 20% of the volume of the aliquot. 
     
     
       12. The method of  claim 2 , further comprising:
 normalizing the one or more probe binding parameters of each sample to account for the reduction in albumin concentration caused by the solvent in the mixture; 
 optionally, where the steps of deriving and normalizing the one or more probe binding parameters are performed simultaneously. 
 
     
     
       13. The method  claim 2 , further comprising:
 normalizing the one or more probe binding parameters of each sample to account for the reduction in serum albumin concentration caused by medical conditions of the patient. 
 
     
     
       14. The method  claim 2 , where the one or more control parameters comprise at least one range of control parameters of control patients. 
     
     
       15. The method of  claim 7 , further comprising:
 repeating the steps of mixing a probe, mixing a solvent, measuring the concentrations, deriving a one or more probe binding parameters for each of two or more aliquots, where a different amount of solvent is used for each of the two or more repetitions; optionally, where the one or more probe binding parameters derived for the two or more repetitions are averaged to derive an average probe binding parameters, and where the averaged one or more probe binding parameters are compared in the step of comparing the one or more probe binding parameters. 
 
     
     
       16. The method of  claim 2 , where the volume of the aliquot is less than 100 μL, and optionally, less than 50 μL. 
     
     
       17. The method of  claim 2 , where the probe is labeled with a spin-label. 
     
     
       18. The method of  claim 2 , where the extracellular fluid includes at least one of: blood serum, blood plasma, lymph fluid, and spinal fluid. 
     
     
       19. The method of  claim 2 , where analyzing each mixture comprises verifying that the mixture has reached binding equilibrium, optionally by:
 measuring two EPR spectra of the mixture at two different times; 
 comparing the two EPR spectra to determine whether intensities of spectral lines (spectral parameters) in the EPR spectra correspond.